Toxoplasma gondii is a zoonotic human parasite with worldwide distribution. Disease in the growing ranks of immunosuppressed patients is primarily due to reactivation of dormant bradyzoite cysts. Development of the bradyzoite form can be studied in vitro, but few molecular or genetic details underlying this important process are known. Taking advantage of a novel multifunctional microarray, the research outlined below will provide a comprehensive genome-wide analysis of bradyzoite formation. Based on preliminary data generated to date, we hypothesize that very early gene products are required for the initiation of bradyzoite differentiation, which will be studied through targeted engineering of transgenic and/or knock-put parasites. Specific Aim 1: Identification of genes that define bradyzoite induction. 1.a. Define expression patterns for putative bradyzoite genes. Multiple induction methods will be employed to ascertain unique bradyzoite-specific genes. 1.b. Focus downstream gene targeting. Using a rational selection process coupled with bioinformatic analysis, genes will be targeted for genetic knock-out experiments. Specific Aim 2: Molecular dissection of critical stage-specific transcripts through genetic perturbation. 2.a. Genetically delete (knock-out) selected genes of interest. Electro-transfection and drug selection will be used to generate allelic knock-out mutants. 2.b. Assess putative bradyzoite induction mutants in vitro. Mutants defective in multiple methods of in vitro bradyzoite formation will identify genes likely to be essential in the differentiation process. 2.c. Verify specific influence of the target gene on bradyzoite induction through fitness assays and complementation. To ensure that phenotypes are directly related to bradyzoite formation, mutants will be complemented and verified for growth and infectivity fitness. The long-term goal of this project is to establish a more complete understanding of the ubiquitous and pathogenic bradyzoite stage of T. gondii. The experiments proposed in this study are expected to lead to the identification of a number of genes involved in the differentiation process in T. gondii. Besides being of interest from a general biological standpoint, such genes should provide novel insights into the development of bradyzoite-specific therapeutics. In addition to increasing my research skills, this may provide key insights leading to the development of drugs designed against bradyzoite targets, or disease-attenuated organisms useful in developing vaccines.